Smooth muscle aldolase C-bound inositol 1,4,5-trisphosphate studied in vitro under physiological conditions.
Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding to aldolase C tetramer (aldolase4) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) under conditions which approximated the in vivo state. Anions were found to have major effects. Decreasing [KCl] from 100 to 10mM, at 0 degrees C and pH 7.0, increased maximal Ins(1,4,5)P3 binding to 1.0 to 2.4mol per mol aldolase4. At 10 and 30mEq/l [Cl-], an additional high affinity site was detected (Kds = 0.43 and 0.86 microM, respectively). Increasing concentrations of other anions (SO42-, propanoate-, HCO3-, acetate-) also inhibited binding, but effects would be minimal at concentrations of these anions present in the cytoplasm of living cells. Ins(1,3,4)P3 displacement of aldolase C-bound Ins(1,4,5)P3 was sensitive to [Cl-]; at 30mEq/l [Cl-] and 37 degrees C, Ins(1,3,4)P3 released 20% of bound Ins(1,4,5)P3 at concentrations of 100nM. Changing temperature from 0 to 37 degrees C increased Kds for Ins(1,4,5)P3 binding. Changes in free [Ca2+], [Mg2+], [Na+] and [K+] and changes in osmolality had no effect on Ins(1,4,5)P3 binding to aldolase C. In vivo Ins(1,4,5)P3-aldolase4 binding at 30mEq/l [Cl-] and 37 degrees C were calculated for different [Ins(1,4,5)P3]free over the range 0.2 to 1.0 microM. For different cytoplasmic [Ins(1,4,5)P3]free. Ins(1,4,5)P3 binding to aldolase4 was sufficient, if acutely released, to nearly double cytoplasmic [Ins(1,4,5)P3]free. We proposed a schema whereby release of aldolase C-bound Ins(1,4,5)P3 evoked by Ins(1,3,4)P3 amplifies effects of phospholipase C-formed Ins(1,4,5)P3.
Baron
C B
CB
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, USA. cbaron@mail.med.upenn.edu
Greeley
P
P
Coburn
R F
RF
D000818:Animals; D000838:Anions / Q000494:pharmacology; D002254:Carbonates / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D013552:Swine; D013696:Temperature; D014132:Trachea / Q000201:enzymology
D000838:Anions; D002254:Carbonates; D007295:Inositol Phosphates; D010710:Phosphates; D012965:Sodium Chloride; D011189:Potassium Chloride; D015544:Inositol 1,4,5-Trisphosphate; C058097:inositol 1,3,4-trisphosphate; C037593:potassium carbonate; D005634:Fructose-Bisphosphate Aldolase; D011188:Potassium